In Vitro Anti-Diabetic Activities and UHPLC-ESI-MS/MS Profile of Muntingia calabura Leaves Extract

Anti-diabetic compounds from natural sources are now being preferred to prevent or treat diabetes due to adverse effects of synthetic drugs. The decoction of Muntingia calabura leaves was traditionally consumed for diabetes treatment. However, there has not been any published data currently available on the processing effects on this plant’s biological activity and phytochemical profile. Therefore, this study aims to evaluate the effect of three drying methods (freeze-drying (FD), air-drying (AD), and oven-drying (OD)) and ethanol:water ratios (0, 50, and 100%) on in vitro anti-diabetic activities of M. calabura leaves. In addition, an ultrahigh-performance-liquid chromatography–electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method was used to characterize the metabolites in the active extract. The FD M. calabura leaves, extracted with 50% ethanol, is the most active extract that exhibits a high α-glucosidase and α-amylase inhibitory activities with IC50 values of 0.46 ± 0.05 and 26.39 ± 3.93 µg/mL, respectively. Sixty-one compounds were tentatively identified by using UHPLC-ESI-MS/MS from the most active extract. Quantitative analysis, by using UHPLC, revealed that geniposide, daidzein, quercitrin, 6-hydroxyflavanone, kaempferol, and formononetin were predominant compounds identified from the active extract. The results have laid down preliminary steps toward developing M. calabura leaves extract as a potential source of bioactive compounds for diabetic treatment.


Introduction
Diabetes mellitus (DM) is a chronic metabolic disorder and an inappropriate hyperglycemia due to either insulin secretion deficiency or a combination of insulin resistance and inadequate insulin secretion to compensate blood glucose levels [1]. While the type 1 is usually associated with the destruction of pancreatic islet β-cell by the autoimmune process, the type 2 diabetes involves a combination of insulin resistance and defective compensatory insulin secretion [2]. In Malaysia, one in five adults, or 18.3% of the population, is estimated to suffer from the implications of DM in 2019, and these numbers are expected to grow, as a spike has been observed from 13.4% diabetes prevalence in a 2015 report [3]. There are six major drug classes that manage hyperglycemia in DM patients, including biguanides (i.e., metformin), thiazolidinediones (i.e., pioglitazone), sulfonylureas (i.e., glimepiride), meglitinides (i.e., repaglinide), dipeptidyl peptidase IV inhibitors (i.e., sitagliptin), and α-glucosidase inhibitors (i.e., acarbose) [1,[4][5][6][7]. However, the naturally occurring anti-diabetic compounds are now gaining popularity as an efficient and wholesome approach toward diabetes management as an alternative compared to synthetic drugs that cause toxic side effects such as hepatic disorders and other negative gastrointestinal symptoms after the consumption over period of time [8,9].
Before commencement of an extraction process, the plant sample is required to undergo a pre-extraction treatment (drying and grinding) to increase the shelf-life, as well as to maximize the extraction of metabolites from the plant [24]. Drying method can be categorized into two types; thermal drying includes the application of heat on the plant sample (hot air-drying, oven-drying (OD), microwave-drying, and sun-drying), while non-thermal drying avoids direct heat to dry the sample (freeze-drying (FD) and air-drying (AD)) [25]. Herbal industries prefers to use oven and AD method in their commercial facilities, as these methods are cost effective and easy to accomplish [26]. The utilization of suitable solvent is one of the key aspects of extraction to give the highest metabolites recovery available in the plant sample. The use of high polarity solvents might affect the solubility of the non-polar compounds, likewise, upon the use of low polarity solvents [27]. The loss of metabolites that are not able to be extracted out may hinder the full capacity of a plant sample to be established as a good antihyperglycemic alternative regimen. Therefore, an efficient extraction process that distills and preserves the indispensable bioactive compounds, contributing to the hypoglycemic effect of a plant extract, is crucial.
Though the hypoglycemic bioactivity of this plant has been previously reported [14], the effects of the drying method and extracting solvent on M. calabura bioactive compounds and, hence, the hypoglycemic activity, has not been discovered. Therefore, this study aims to investigate the optimal combination of drying method and extracting solvents (ethanol:water) with appropriate ratio that can produce M. calabura extracts with the most desirable and potent in vitro anti-diabetic activities. In addition, an ultrahighperformance-liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method was used to characterize the metabolites of the active extract, based on their MS/MS data, for the first time. The results obtained from this study will allow recommendations on the best combination of drying method and extracting solvent to obtain M. calabura extract with significant potential in selected bioactivity and high productivity.

Influence of Varied Drying Process and Ethanol Concentrations on Anti-Diabetic Activity
The bioactivity results of M. calabura leaves extracts, from different drying methods and ethanol:water ratios, are shown in Table 1. M. calabura leaves extracts were tested in two in vitro anti-diabetic assays, named α-glucosidase and α-amylase inhibition activity. In these two activities, the hypoglycemic potential of M. calabura extracts, to reduce the blood sugar level through competitive binding to the active site of the enzyme to prevent the conversion of carbohydrates to simple glucose, were determined [28]. The α-glucosidase inhibitory activity was designed to assess the ability of an extract to suppress the active enzyme from catalyzing the conversion of glucose from the disaccharides, which occurs in the small intestine [29]. In this study, PNPG, a specific substrate that allows hydrolyzation to 4-nitrophenol (yellow colored product) by α-glucosidase enzyme, quantitates at a maximum wavelength of 405 nm [30], while α-amylase inhibitory activity was used to measure the free carbonyl group of the reducing sugar (maltose) converted by the α-amylase from the complex carbohydrates (potato starch) [31]. The aldehyde group from the maltose reduces the yellow colored DNS to form 3-amino-5-nitrosalicylic acid (brick red colored solution) [31]. The concentration of maltose presence in the sample, determine the intensity of the color at a wavelength of 540 nm [32]. Table 1. α-Glucosidase and α-amylase inhibitory activity of M. calabura leaves dried with oven-drying (OD), air-drying (AD), and freeze-drying (FD) and extracted with 0, 50, and 100% ethanol.  50 values of five biological replicates are expressed as means ± standard deviation. The uppercase letter is used to demonstrate the different ethanol:water ratio for the same drying process, while the lowercase letter is used to demonstrate the varied drying process for the same ethanol:water ratio. The different superscript letters represent significant differences at p < 0.05 between samples.
The results demonstrated in Table 1 show the IC 50 value of the α-glucosidase inhibition activity of M. calabura leaves extracts, ranging from 0.46-2.76 µg/mL in comparison to quercetin (2.15 ± 0.26 µg/mL), while for α-amylase inhibition activity, the IC 50 value, ranging from 23.84-185.17 µg/mL in comparison to acarbose (0.68 ± 0.14 µg/mL). In α-glucosidase inhibition activity, the effect of different ethanol:water ratios played the most significant role that affected α-glucosidase inhibition ability of M. calabura leaves compared to the different drying methods. The 50 and 100% ethanolic extracts from FD and AD leaves showed the lowest IC 50 value with no significant difference (p > 0.05), ranging from 0.46 ± 0.05 to 1.07 ± 0.06 µg/mL. On the contrary, the IC 50 value of 50% ethanolic extract from OD leaves was the lowest compared to that of the 100 and 0% ratios from the same drying method, whereas the 0% ethanolic extract from all the drying methods showed significantly lower α-glucosidase inhibition potential compared to that of other extracts. As for the α-amylase inhibition activity, the lowest IC 50 value was found in the 50% ethanolic extract, regardless of its drying method, and in the 100% ethanolic extract from the FD method. When comparing the different drying methods, the FD M. calabura leaves showed the highest α-amylase inhibition capability compared to the AD and OD methods in both 50 and 100% ethanolic extracts. However, the OD leaves showed the lowest IC 50 value when extracted with 0% ethanol. The use of 50% ethanol, evidently, was the best ethanol:water ratio to extract out the phytochemical constituents with hypoglycemic potential in M. calabura leaves for both tested bioactivities. In line with the previous in vitro study, where the use of alcohol and water mixture could efficiently extract out the wider spectrum of phytochemicals constituents that might contribute to the bioactivity as compared to a mono-component solvent system [33]. Besides that, the utilization of the 50% ethanolic ratio allows utilization of less organic solvent, increasing extract's solubility in less toxic organic environment and confirming its usage harmlessly in in vivo studies [34].
In this study, FD and AD extracts were procured as the most effective drying methods in order to identify the α-glucosidase and α-amylase inhibitors followed by OD extraction. The absence of thermal degradation hindered the degradative enzyme to function [35]. Several studies have concurred that the FD method enables higher retention of phytochemical compounds that leads to enhanced biological activity of the plant [36]. In contrast, the thermal processing such as oven-drying, hot air-drying, and microwave-drying impeded the extraction of phytochemicals of the plant by thermal breakdown, disrupting the integrity of cell structure and migration of components, catalyzing further disruption by various chemical reactions such as enzymes, light, and oxygen [37]. In addition, the utilization of the FD method will shorten the extraction process by expediting the drying time, compared to the AD technique, as well as potentially providing higher yield of anti-diabetic phytochemical constituents [38,39]. Hence, the 50% ethanol:water ratio on FD M. calabura leaves, with strong α-glucosidase and α-amylase inhibitory activity, can be potentially exploited further to create a natural regimen from M. calabura leaves to aid in the blood sugar management of a diabetic patient.

UHPLC-ESI-MS/MS Characterization of Phytoconstituents in the M. calabura Leaves Extract
Based on the anti-diabetic activity of M. calabura leaves, the FD 50% ethanolic extract was identified and selected as the most active extract. This extract was then subjected to UHPLC-ESI-MS/MS identification to have better insight on the constituents present that might enhance the bioactivities. Figure 1 shows the total ion chromatogram and UV-Vis chromatogram (254 nm) of the FD 50% ethanolic M. calabura leaves extract for both positive and negative ion mode. A total of 61 compounds (Table 2) were putatively identified based on their molecular ions in the full scan mass spectra, retention time, fragmentation patterns, mass error, and UV absorption spectra supported with database mining, such as MassBank databases (https://massbank.eu/MassBank/), Human Metabolomics Databases (HMDB) (https://hmdb.ca/), Metabolomics Workbench databases (https://www.metabolomicsworkbench.org/), Chemspider databases (http: //www.chemspider.com/), and MassFrontier (version 8.0) software (Thermo Scientific, Waltham, MA, USA). The compounds identified include five catechin derivatives, five types of kaempferol derivatives, three types of quercetin derivatives, one apigenin derivative compound, three types of luteolin derivatives, seven types of daidzein derivatives, 19 types of other flavonoids, two types of anthocyanin compounds, two types of chalcone compounds, six types of quinone derivatives, two types of lactones derivatives, two types of alkaloid derivatives, two types of sugar, one terpene glycoside, and one ellagitannins derivative.  [40]. Figure 2 [40].     [41]. This compound was previously isolated and characterized in this plant by Nshimo et al. [42]. deprotonated molecule [M-H] at m/z 1187.2678. This compound exhibited the same fragmentation patterns in the MS/MS analysis as buddlenoid A, with double of its monomer [41]. Another kaempferol derivative was also identified in the chromatogram at tR = 13.63 min, with a deprotonated molecule [M-H] − at m/z 287.0539. Additionally, m/z 271.0607 [M-H-16] − occurs due to loss of CH4 in the MS/MS analysis, while m/z 269.0723, 216.9894, and 119.0077 are typical fragments for kaempferol derivatives [40]. Hence, this compound was identified as dihydrokaempferol (Peak 37).     [40]. Figure 6 shows the main fragmentation pathways found in luteolin derivatives.    [40]. Figure 6 shows the main fragmentation pathways found in luteolin derivatives.

Luteolin Derivatives
One luteolin derivative named velutin (Peak 40) was tentatively identified, as they share three significant product ions [M-H] − at m/z 285.0284, 255.0299, 227.0350, and 213.0395, which were the base peaks for luteolin aglycone [40]. Velutin and its isomers (Peak 40, 45, and 46) were identified based on its deprotonated molecule [M-H] − at m/z 313.0719 at t R = 14.03, 15.01, and 15.19 min [40]. Figure 6 shows the main fragmentation pathways found in luteolin derivatives.  [44]. Then, 3 -hydroxydaidzein was previously isolated and characterized in this plant by Matsuda et al. [45]. While at t R = 16.13 and 16.29, formononetin (Peak 58) and its isomer (Peak 59) were identified based on its protonated molecule [M+H] + at m/z 269.0819 and the four fragmentation ions at MS/MS analysis that agreed to the base peak of daidzein [40]. The identification of this compound was confirmed with the authentic standard.  Figure 7 at tR = 14.38 and 14.78 min, respectively [44]. Then, 3′-hydroxydaidzein was previously isolated and characterized in this plant by Matsuda et al. [45]. While at tR = 16.13 and 16.29, formononetin (Peak 58) and its isomer (Peak 59) were identified based on its protonated molecule [M+H] + at m/z 269.0819 and the four fragmentation ions at MS/MS analysis that agreed to the base peak of daidzein [40]. The identification of this compound was confirmed with the authentic standard.     [40] t R = retention time; MF = molecular formula; * Indicate the compounds previously isolated from M. calabura; # indicated the compounds that were confirmed with the authentic standard.

UHPLC Absolute Quantification
Six standard compounds named geniposide, daidzein, quercitrin, kaempferol, formononetin, and 6-hydroxyflavanone that were previously identified by using UHPLC-ESI-MS/MS technique, were subjected to UHPLC quantification to determine the absolute amount of each standard in the most active extract. The result in Table 3 shows the metabolites content in FD M. calabura leaves extracted with 50% ethanol, ranging from 56.58 ± 0.28 to 650.01 ± 0.12 µg/mg of extract. The highest contents of metabolites in the extract were in the following order geniposide, daidzein, quercitrin, 6-hydroxyflavanone, kaempferol, and formononetin. Each of these metabolites may contribute to the anti-hyperglycemic effect of M. calabura leaves. For example, geniposide plays an important role in reducing glycogenolysis process through impairment of hepatic glycogen phosphorylase and glucose-6-phosphatase activity, as well as their protein expression in high fat diet-streptozotocin (STZ) induced diabetic mice [18]. On the other hand, daidzein helps to increase the mRNA level in the β-cells to exert more insulin production, as well as restoring the glucose metabolic enzyme activities in daidzein-supplemented non-obese diabetic mice [19]. In addition, one of the quercetin derivatives has proven to have a protective effect on the intact β-cells of the islets of Langerhans by inhibiting lipid peroxidation, as well as its ability to scavenge free radicals from STZ-induced oxidative stress in STZ-induced diabetic rats [20]. Although no research has been conducted regarding the proficiency of 6-hydroxyflavanone on directly reducing the blood glucose level, there are some studies that have demonstrated the electron donating ability of 6-hydroxyflavanone to scavenge free radicals induced by 2,2-diphenyl-1-picrylhydrazyl in in vitro antioxidant study [21,59]. Besides that, kaempferol, another flavonoid-type compound that was identified and quantified, also reportedly shows hypoglycemic effect by lowering the levels of glycoprotein in the liver and increment of insulin production, as well as enhancement of glucose utilization in STZ-induced rats [22]. Formononetin helps in the activation of peroxisome proliferatoractivated receptor (PPAR-γ) genes in liver, which, in turn, regulates the blood glucose level, the cell surface receptor protein (FAS) may not induce the cleavage of SREBP-1C site in stimulating lipid synthesis due to an antagonistic interaction between formononetin and PPAR-γ genes [23]. Therefore, these identified metabolites were quantified based on its positive attributes in lowering the blood glucose level, either through in vivo or in vitro study, that could potentially influenced the hypoglycemic aptitude of M. calabura leaves.

Plant Materials
The fresh M. calabura were harvested from Universiti Putra Malaysia's Mosque. Number of voucher specimen (SK 3345/18) was deposited by a botanist (Dr. Mohd Firdaus Ismail) in the herbarium of Institute of Bioscience, Universiti Putra Malaysia. The leaves of five different trees of M. calabura were harvested and separated into three drying processes, namely oven-drying (OD), air-drying (AD), and freeze-drying (FD). The fresh leaves were assigned at 40 • C in a convention oven (Protech, Seri Kembangan, Malaysia), for a duration of 10 days for OD processed leaves. On the other hand, for the AD sample, the leaves were dried at 37 • C for the duration of 2 weeks. As for the FD sample, the leaves were stored for 4 days in −80 • C freezer (Haier, Surrey, UK) prior to 3 days FD (LabConco, Kansas City, MO, USA). All samples were confirmed to absolute dryness, as their constant weight was achieved before grinding process started. The ground samples were then stored at 2-7 • C in airtight container until further analysis. Total 5 g of each ground leaves was soaked and sonicated in 100 mL of 0, 50, and 100% ethanol:water ratio, respectively, for 60 min by using ultrasonic bath sonicator (Kudos, Shanghai, China). The Whatman filter paper no. 1 was used to filter the mixture and the filtered mixture was concentrated under controlled temperature at 40 • C by using a rotatory evaporator (Buchi LaboratoriumsTechnik, Flawil, Switzerland). To ensure complete dryness, all extracts were then lyophilized in a freeze dryer (LabConco, Kansas City, MO, USA) and subsequently stored at −20 • C until further analysis.

α-Glucosidase Inhibition Assay
The α-glucosidase inhibition activity assay has been conducted according to the demonstrated method with modifications [60]. The PNPG in 50 mM phosphate buffer (pH 6.5) has been used as the substrate, which was comparable to the intestinal fluid. Next, sample extracts were prepared at 200 µg/mL and 6 serial dilutions were done. In the 96-well plate, a reaction consisting of 130 µL of 30 mM phosphate buffer, 10 µL of enzyme, and 10 µL of extracts was incubated at 37 • C for 5 min. Then, 50 µL of substrate was added in the reaction and was further incubated at 37 • C for another 15 min. Next, 50 µL of 2 M glycine (pH 10) was then added to stop the reaction. The absorbance was measured by using a spectrophotometer (Spectramax PLUS, San Jose, CA, USA) at 405 nm wavelength. The α-glucosidase inhibitory activity was calculated using the equation; [(An − As)/An] × 100%, where An is the difference in absorbance of the negative control and all the blanks, and As is the difference in absorbance of the sample and all the blanks. The α-glucosidase inhibitory activity was expressed as IC 50 value (µg/mL) to represent M. calabura extract concentration that was needed to inhibit the enzyme activity by 50%. Quercetin was tested and used as positive control.

α-Amylase Inhibition Assay
The α-amylase inhibition activity assay was carried out based on the demonstrated method with modifications [32]. In a 96-well plate, a reaction mixture containing 50 µL of 100 mM phosphate buffer at pH 6.8, 10 µL of 2 U/mL porcine α-amylase, and 20 µL of varying concentrations of the extract (0.04, 0.07, 0.14, 0.28, and 0.56 mg/mL) was incubated at 37 • C for 20 min. Then, 20 µL of substrate (1% starch dissolved in 100 mM phosphate buffer at pH 6.8) was added, and the mixture was further incubated at 37 • C for 30 min. The reaction was then stopped by adding 100 µL of the DNS color reagent and boiled for 10 min. The absorbance was measured by using a spectrophotometer (Spectramax PLUS, San Jose, CA, USA) at 540 nm wavelength. The α-amylase inhibitory activity has been calculated using the equation; [(An − As)/An] × 100%, where An is the difference in absorbance of the negative control and all the blanks, and As is the difference in absorbance of the sample and all the blanks. The α-amylase inhibitory activity was expressed as IC 50 (µg/mL). Acarbose was tested and employed as a positive control in each plate of α-amylase activity.

UHPLC-ESI-MS/MS Analysis
The sample preparation for UHPLC-ESI-MS/MS analysis was prepared based on the reported method with modifications [36]. The most active extract (4 mg) was dissolved in 2 mL of LCMS-grade methanol and filtered through a 0.22 µm filter prior to 20.0 µL injection to the UHPLC-ESI-MS/MS analysis. The analysis was performed using an Dionex Ultimate 3000 UHPLC coupled with Q Exactive™ Focus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a binary pump, vacuum degasser, temperature-controlled autosampler, diode array detector (200-600 nm range; 5 nm bandwidth), and a heated electrospray ionization source. The separation was conducted on a Waters Acquity UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm) (Waters Corp, Milford, MA, USA). The gradient solvent system was then commenced from 95:5-0:100 (v/v) of water with 0.1% formic acid: acetonitrile with 0.1% formic acid over 39 min, with a flow rate of 0.25 mL/min. Negative and positive ion modes were acquired and recorded on Q Exactive™ Focus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The MSn analytical conditions were as follows: spray volt-pressure −3.6 kV; equipment temperature, 37.8 • C; capillary temperature, 300 • C; auxiliary gas at 8 units; sheath gas at 50 units; scan range m/z 150-1200, data acquisition frequency at 12 Hz, and collision-induced dissociation energy was adjusted to 30%. The data was recorded and processed using the Thermo Xcalibur Qual Browser software 4.0 (Thermo Fisher Scientific, Bremen, Germany).

Absolute Quantification from FD Leaves Extracted with 50% Ethanol by UHPLC
The most active extract was also subjected to absolute quantification for several phenolics named daidzein, quercitrin, kaempferol, formononetin, and 6-hydroxyflavanone, as well as a terpene glycoside, geniposide. The sample preparation for UHPLC analysis was according to the reported method with slight adjustment [36]. The active extract (10 mg) of the M. calabura was dissolved in 2 mL of LCMS-grade methanol and filtered through a 0.22 µm filter prior to 2.0 µL injection to the UHPLC analysis. The analysis was performed using a Waters Acquity UHPLC (Waters Corp, Milford, MA, USA) equipped with a binary pump, vacuum degasser, temperature-controlled autosampler, column heater, and 2998 photodiode array (PDA) detector (200-600 nm range; 5 nm bandwidth). The separation was conducted on a Waters Acquity UPLC HSS T3 column (1.8 µm, 2.1 × 150 mm) (Waters Corp, Milfor, MA, USA) with the same gradient solvent system mentioned in profiling section, where the gradient solvent system was commenced from 95:5-0:100 (v/v) of water with 0.1% FA:acetonitrile with 0.1% FA over 39 min with a flow rate of 0.25 mL/min. The detection wavelength was set at 254 nm. The data was evaluated and processed by using the Waters Empower Chromatography Data System software. The metabolite contents in the plant extract were calculated, based on the area under the peak, and expressed as µg/mg of plant extract.
The quantification method was validated according to International Council of Harmonization (ICH) tripartite guidelines [61] based on the following criteria: • Specificity: the retention time of the standards and extract are complementary with no contaminants or impurities detected in the eluent, with the equal volume (2.0 µL) of sample, standards, and solvent injected into the chromatography system. • Repeatability precision: all the relative standard deviation (RSD) were <2%, indicating high precision. The repeatability precision test was acquired by three-times injections at three concentration levels (5, 20, and 40 µg/mL) for each standard (daidzein, quercitrin, kaempferol, formononetin, 6-hydroxyflavanone, and geniposide). • Linearity and range: the calibration curve was obtained by three data points at 5, 20, and 40 µg/mL. Each calibration curve was determined by the averaging triplicate value of each concentration. Table 4 shows the concentration range, regression equation, and correlation coefficient (R 2 ). where SD is the standard deviation of the response, and M is the slope of calibration curve. Table 4 demonstrates the LOD and LOQ value for the six tested standards.

Statistical Analysis
The results of the five biological replicates were expressed in mean ± standard deviation. The statistical analysis was done by using Minitab software (Version 16, Minitab Inc, State College, PA, USA), while the significant difference in the results was determined by analysis of variance (ANOVA) with post hoc Tukey pairwise multiple-comparisons test (p < 0.05, significant).

Conclusions
In conclusion, M. calabura leaves, extracted with 50% ethanol and dried by using FD method, were revealed to be the best condition to extract the anti-diabetic metabolites. A total of sixty-one compounds were tentatively identified in the active extract by using UHPLC-ESI-MS/MS technique, including five catechin derivatives, five types of kaempferol derivatives, three types of quercetin derivatives, one apigenin derivative compound, three types of luteolin derivatives, seven types of daidzein derivatives, nineteen types of other flavonoids, two types of anthocyanin compounds, two types of chalcone compounds, six types of quinone derivatives, two types of lactones derivatives, two types of alkaloid derivatives, two types of sugar, one terpene glycoside, and one ellagitannins derivative. The highest contents of metabolites in the active extract were in the following order: geniposide, daidzein, quercitrin, 6-hydroxyflavanone, kaempferol, and formononetin. Hence, this study suggests that, with the incorporation of the FD method, M. calabura leaves when extracted with 50% ethanol, can play a significant role as a potential for the development of naturally derived herbal medicinal components that not only help to inhibit diabetes related complications but also impedes toxic side effects of synthetic anti-diabetic drugs.